Abstract The dysregulation of the metabolic pathways is the direct cause of inborn errors of metabolism and also leads to common diseases like cancers and diabetes. The applicant's long-term objective is to develop an integrated strategy combining computation, CRISPR genome editing and metabolomics to study the poorly characterized metabolic pathways underlying human diseases. The results of these studies will generate novel hypotheses for diagnosing metabolic diseases of unknown causes and provide alternative directions for disease interventions. The applicant has previously obtained rigorous graduate training in biochemistry and cell biology, including membrane lipid biology. During the ongoing NRSA F32 postdoctoral funding period, the applicant has developed an integrated approach to study a mitochondrial enzyme of unknown function, CLYBL, and revealed its function in regulating mitochondrial vitamin B12 (B12)-dependent processes. This finding mirrors previous human genetic studies that associate loss-of-function of CLYBL with low circulating B12 levels. For the K99/R00 application, the applicant proposes to focus on two mitochondria-localized, essential metabolic pathways in human: (1) to identify missing regulators of the mitochondrial B12 pathway; (2) to perform loss-of-function studies of the mitochondrial type II fatty acid synthesis (mtFASII). To achieve these goals, two major strategies will be applied: (a) to leverage genome-wide computational approaches and publicly available databases to predict new pathway regulators; (b) to combine CRISPR editing in cultured cells and high-resolution LC- MS based metabolomics (including lipidomics) and proteomics (including top-down proteomics) to probe metabolism. The applicant's host laboratory and institute provide an ideal training environment for the proposed research. Her postdoctoral mentor Dr. Vamsi Mootha's laboratory has previously developed genome-wide computational methods to predict the mitochondrial proteome ? the same toolset that could predict novel metabolic regulators. The laboratory is also an early adopter of metabolomics and part of the Broad Institute Metabolism Program. The applicant has obtained initial training in LC-MS methods to profile polar metabolites. And during the K99/R00 funding period, she will receive additional training in lipidomics and advanced native proteomics method to study the fatty acid acyl-chain extension during the mtFASII. Successful completion of this project will provide fundamental insights into mitochondrial cofactor metabolism and regulation of lipid homeostasis, and might introduce new directions for diagnosing metabolic diseases. Meanwhile, the career development plan will prepare the applicant to transition into an independent investigator in the field of metabolism.